Biologically active substances of plant origin: Methods of extraction and analysis

Biologically active substances (BAS) of plant origin play a key role in the development of functional foods, bio-additives and medicines. Their use, however, is restricted by difficulties in extraction and low stability. This paper is devoted to investigation of the structure, characteristics and biological impact of the main BAS classes — phenolic compounds, among which flavonoids, stilbenes and lignans stand out. Their role as potent antioxidants and anti-inflammatory agents, as well as their effect on the health of the cardiovascular system, is emphasized. The review covers both traditional and modern methods for extraction of biologically active components from plants, such as maceration, distillation, extraction by the Soxhlet method, ultrasound, microwave and supercritical technologies of extraction. The authors describe in detail spectrophotometric methods DPPH, ABTS, as well as fluorescent method ORAC and its improved version ORAC-SIA, intended for the assessment of the total antioxidant capacity of individual compounds and their combination upon the physiologically relevant values of the acidity of the environment. The results of the study demonstrate the dependency of the BAS yield on the solvent type, hydromodule and a degree of comminution of raw materials. Extraction parameters such as temperature, pressure and pH significantly affect the preservation of thermolabile compounds. It has been found that modification of the structure of cyclotides increases the bioavailability of peptide preparations by 1.5–2 times. Analytical methods like ORAC and its modification ORAC-SIA allows for assessing the antioxidant activity of BAS in complex mixtures including food products. Based on the performed analysis, the conclusions were made regarding the need for optimization of “green” extraction methods, development of the standardized protocols of analysis and in-depth study of the interrelation “structure–activity” for targeted design of BAS.

1. Carpena, M., da Pereira, R., Garcia-Perez, P., Otero, P., Soria-Lopez, A., Chamorro, F. et al. (2022). An Overview of Food Bioactive Compounds and Their Properties Chapter in a book: Membrane Separation of Food Bioactive Ingredients Springer, Cham, 2022. https://doi.org/10.1007/978-3-030-84643-5_2

2. Ferdes, M. (2018). Antimicrobial compounds from plants. Chapter in a book Fighting Antimicrobial Resistance. IAPC Publishing, Zagreb, Croatia, 2018. https://doi.org/10.5599/obp.15.15

3. Rawat, P., Singh, Y., Bisht, M., Pal, M. (2023). Modern Analytical Techniques for Extraction, Purification, and Structural Characterization of Microbial Bioactive Compounds. Chapter in a book: Microbial Bioactive Compounds. Springer, Cham, 2023. https://doi.org/10.1007/978-3-031-40082-7_5

4. Daliri, E. B. -M., Lee, B. H., Oh, D. H. (2017). Current trends and perspectives of bioactive peptides. Critical Reviews in Food Science and Nutrition, 58(13), 2273– 2284. https://doi.org/10.1080/10408398.2017.1319795

5. Mollica, A., Costante, R., Stefanucci, A., Novellino, E. (2015). Cyclotides: A natural combinatorial peptide library or a bioactive sequence player? Journal of Enzyme Inhibition and Medicinal Chemistry, 30(4), 575–580. https://doi.org/10.3109/14756366.2014.954108

6. Rahman, M. M., Rahaman, M. S., Islam, M. R., Rahman, F., Mithi, F. M., Alqahtani, T. et al. (2022). Role of phenolic compounds in human disease: Current knowledge and future prospects. Molecules, 27(1), Article 233. https://doi.org/10.3390/molecules27010233

7. Zhang, Y., Cai, P., Cheng, G., Zhang, Y. (2022). A brief review of phenolic compounds identified from plants: Their extraction, analysis, and biological activity. Natural Product Communications, 17(1), 1–14. https://doi.org/10.1177/1934578X211069721

8. Kapustin, M. A., Chubarova, A. S., Halavatch, T. N., Cigankov, V. G., Bondaruk, A. M., Kurchenko, V. P. (2016). Methods of active compounds with cyclic oligosaccharides nanocomplexes obtaining, analysis of it physical and chemical properties and use in food production. Proceedings of the Belarusian State University. Series of Physiological, Biochemical and Molecular Biology Sciences, 11(1), 73–100. (In Russian)

9. Tanase, C., Coșarcă, S., Muntean, D.-L. (2019). A critical review of phenolic compounds extracted from the bark of woody vascular plants and their potential biological activity. Molecules, 24(6), Article 1182. https://doi.org/10.3390/molecules24061182

10. Mendonça, E. L. S. S., Xavier, J. A., Fragoso, M. B. T., Silva, M. O., Escodro, P. B., Oliveira, A. C. M. et al. (2024). E-Stilbenes: General chemical and biological aspects, potential pharmacological activity based on the Nrf2 pathway. Pharmaceuticals, 17(2), Article 232. https://doi.org/10.3390/ph17020232

11. Bonnefont-Rousselot, D. (2016). Resveratrol and cardiovascular diseases. Nutrients, 8(5), Article 250. https://doi.org/10.3390/nu8050250

12. Braun, C., Dohlen, S., Ilg, Y., Brodkorb, F., Fischer, B., Heindirk, P. et al. (2017). Antimicrobial activity of intrinsic antimicrobial polymers based on poly((tertbutyl-amino)-methyl-styrene) against selected pathogenic and spoilage microorganisms relevant in meat processing facilities. Journal of Antimicrobial Agents, 3(1), Article 136. https://doi.org/10.4172/2472-1212.1000136

13. Al-Khayri, J. M., Sahana, G. R., Nagella, P., Joseph, B. V., Alessa, F. M., Al-Mssallem, M. Q. (2022). Flavonoids as potential anti-inflammatory molecules: A review. Molecules, 27(9), Article 2901. https://doi.org/10.3390/molecules27092901

14. Tian, C., Liu, X., Chang, Y., Wang, R., Lv, T., Cui, C. et al. (2021). Investigation of the anti-inflammatory and antioxidant activities of luteolin, kaempferol, apigenin, and quercetin. South African Journal of Botany, 137, 257–264. https://doi.org/10.1016/j.sajb.2020.10.022

15. Ku, Y.-S., Ng, M.-S., Cheng, S.-S., Lo, A. W.-Y., Xiao, Z., Shin, T.-S. et al. (2020). Understanding the composition, biosynthesis, accumulation, and transport of flavonoids in crops for the promotion of crops as healthy sources of flavonoids for human consumption. Nutrients, 12(6), Article 1717. https://doi.org/10.3390/nu12061717

16. Vinogradova, M. G. (2021). UV spectral analysis of plant raw materials may lily of the valley. Vestnik of Tver State Technical University. Series «Building. Electrical Engineering and Chemical Technology», 2(10), 95–102. (In Russian) https://doi.org/10.46573/2658–7459–2021–95–102

17. Ražná, K., Nôžková, J., Vargaová, A., Harenčár, L., Bjelková, M. (2021). Biological functions of lignans in plants. Agriculture, 67(3), 155–165. https://doi.org/10.2478/agri-2021-0014

18. Kapustin, M. A., Chubarova, H. S., Lodygin, A. D., Rzhepakovsky, I. V., Dudchik, N. V., Tsygankow V. G. et al. (2025). Technology for nanocomplexes of curcuminoids with cyclodextrins production, investigation of their properties and biological activity. Experimental Biology and Biotechnology, 1, 24–39. (In Russian)

19. Gori, A., Boucherle, B., Rey, A., Rome, M., Fuzzati, N., Peuchmaur, M. (2021). Development of an innovative maceration technique to optimize extraction and phase partition of natural products. Fitoterapia, 148, Article 104798. https://doi.org/10.1016/j.fitote.2020.104798

20. Hidayat, R., Wulandari, P. (2021). Methods of extraction: Maceration, percolation, and decoction. Eureka Herba Indonesia, 2(1), 68–74. https://doi.org/10.37275/ehi.v2i1.15

21. Opitz-Kreher, K., Huber, J. (2023). Twelve Essential Oils of the Bible: Ancient Healing Oils and Their Contemporary Uses. Simon and Schuster, 2023.

22. Stratakos, A. C., Koidis, A. (2016). Methods for Extracting Essential Oils. Chapter in a book: Essential Oils in Food Preservation, Flavor and Safety. Academic Press, 2016. https://doi.org/10.1016/B978-0-12-416641-7.00004-3

23. Azmir, J., Zaidul, I. S. M., Rahman, M. M., Sharif, K. M., Mohamed, A., Sahena, F. et al. (2013). Techniques for extraction of bioactive compounds from plant materials: A review. Journal of Food Engineering, 117(4), 426–436. https://doi.org/10.1016/j.jfoodeng.2013.01.014

24. Tăbărașu, A.-M., Nenciu, F., Anghelache, D.-N., Vlăduț, V.-N., Găgeanu, I. (2024). Hybrid percolation–ultrasound method for extracting bioactive compounds from Urtica dioica and Salvia officinalis. Agriculture, 14(9), Article 1561. https://doi.org/10.3390/agriculture14091561

25. Dubashinskaya, N. V., Khishova, O. M., Shimko, O. M. (2007). Characteristics of methods of extract production and their standardization (part II). Pharmacy Herald, 36(2), 70–79. (In Russian)

26. Soxhlet, F. (1879). Die gewichtsanalytische Bestimmung des Milchfettes. Dingler’s Polytechnisches Journal, 232, 461–465. (In German)

27. de Castro, M. D. L., Priego-Capote, F. (2010). Soxhlet extraction: Past and present panacea. Journal of Chromatography A, 1217(16), 2383–2389. https://doi.org/10.1016/j.chroma.2009.11.027

28. Naviglio, D., Scarano, P., Ciaravolo, M., Gallo, M. (2019). Rapid solid-liquid dynamic extraction (RSLDE): A powerful and greener alternative to the latest solid-liquid extraction techniques. Foods, 8(7), Article 245. https://doi.org/10.3390/foods8070245

29. Khan, S., Aslam, R., Makroo, H. (2018). High pressure extraction and its application in the extraction of bioactive compounds: A review. Journal of Food Process Engineering, 42(1), Article e12896. https://doi.org/10.1111/jfpe.12896

30. Herrero, M., Mendiola, J. A., Cifuentes, A., Ibáñez, E. (2010). Supercritical fluid extraction: Recent advances and applications. Journal of Chromatography A, 1217(16), 2495–2511. https://doi.org/10.1016/j.chroma.2009.12.019

31. Dumitrash, P. G., Bologa, M. K., Shemyakova, T. D. (2016). Ultrasound-assisted extraction of biologically active substances from tomato seeds. Surface Engineering and Applied Electrochemistry, 52(3), 270–275. https://doi.org/10.3103/S1068375516030054

32. Wang, C., Zhang, W., Liao, Y., Ye, J., Xu, F., Wang, Q. (2025). Ginkgo biloba flavonoids: Analysis of functions, regulatory mechanisms, and extraction. Plant Biology, 27(6), 962–974. https://doi.org/10.1111/plb.70054

33. Routray, W., Orsat, V. (2012). Microwave-assisted extraction of flavonoids: A review. Food and Bioprocess Technology, 5(3), 409–424. https://doi.org/10.1007/s11947-011-0573-z

34. Naliyadhara, N., Kumar, A., Girisa, S., Daimary, U. D., Hegde, M., Kunnumakkara, A. B. (2022). Pulsed electric field (PEF): Avant-garde extraction escalation technology in food industry. Trends in Food Science and Technology, 122, 238– 255. https://doi.org/10.1016/j.tifs.2022.02.019

35. Sharma, H. P., Patel, H., Sugandha (2017). Enzymatic added extraction and clarification of fruit juices — A review. Critical Reviews in Food Science and Nutrition, 57(6), 1215–1227. https://doi.org/10.1080/10408398.2014.977434

36. Verep, D., Ates, S., Karaoğul, E. (2023). A review of extraction methods for obtaining bioactive compounds in plant-based raw materials. Bartın Orman Fakültesi Dergisi, 25(3), 492–513.

37. Singh, S., Verma, D. K., Thakur, M., Tripathy, S., Patel, A. R., Shah, N. et al. (2021). Supercritical fluid extraction (SCFE) as green extraction technology for highvalue metabolites of algae, its potential trends in food and human health. Food Research International, 150(Part A), Article 110746. https://doi.org/10.1016/j.foodres.2021.110746

38. Matsumoto, H., Nakamura, Y., Hirayama, M., Yoshiki, Y., Okubo, K. (1999). Antioxidant activity of black currant anthocyanin aglycons and their glycosides measured by chemiluminescence in a neutral pH region and in human plasma. Journal of the Science of Food and Agriculture, 50(18), 5034–5037. https://doi.org/10.1021/jf020292i

39. Kurkin, V. A., Ryazanova, T. K., Kurkina, A. V., Egorova, A. V. Method for measuring anthocyanes in crude drugs. Patent RF, no. 2554002, 2015. (In Russian)

40. Eftekhari, M., Alizadeh, M., Ebrahimi, P. (2012). Evaluation of the total phenolics and quercetin content of foliage in mycorrhizal grape (Vitis vinifera L.) varieties and effect of postharvest drying on quercetin yield. Industrial Crops and Products, 38, 160–165. https://doi.org/10.1016/j.indcrop.2012.01.022

41. Ponomarev, S. V., Zotov, A. N., Gvozdkova, O. N., Maltstv, A. Yu., Voronov, D. V. (2024). Phytochemical components and industrial application of grape seeds. A brief review of world research. Efficient Animal Husbandry, 1(191), 70–73. (In Russian)

42. Houldsworth, A. (2024). Role of oxidative stress in neurodegenerative disorders: A review of reactive oxygen species and prevention by antioxidants. Brain Communications, 6(1), Article fcad356. https://doi.org/10.1093/braincomms/fcad356

43. Wang, D., Xiao, H., Lyu, X., Chen, H., Wei, F. (2023). Lipid oxidation in food science and nutritional health: A comprehensive review. Oil Crop Science, 8(1), 35–44. https://doi.org/10.1016/j.ocsci.2023.02.002

44. Pisoschi, A. M., Pop, A. (2015). The role of antioxidants in the chemistry of oxidative stress: A review. European Journal of Medicinal Chemistry, 97, 55–74. https://doi.org/10.1016/j.ejmech.2015.04.040

45. Cordiano, R., Di Gioacchino, M., Mangifesta, R., Panzera, C., Gangemi, S., Minciullo, P.L. (2023). Malondialdehyde as a potential oxidative stress marker for allergy-oriented diseases: An update. Molecules, 28(16), Article 5979. https://doi.org/10.3390/molecules28165979

46. Jiménez-Morales, W. A., Cañizares-Macias, M. D. P., Pedraza-Chaverri, J. (2022). Fast ORAC-SIA method for antioxidant capacity determination in food samples. Food Chemistry, 384, Article 132524. https://doi.org/10.1016/j.foodchem.2022.132524

47. Yashin, A. Ya., Vedenin, A. N., Nemzer, B. V., Yashin, Ya. I. (2019). Berries: Chemical composition, antioxidant activity. Impact of consumption of berries on health of the person. Analytics, 9(3), 222–231. (In Russian) https://doi.org/10.22184/2227-572X.2019.9.3.222.230

48. Apak, R., Gorinstein, S., Böhm, V., Schaich, K. M., Özyürek, M., Güçlü, K. (2013). Methods of measurement and evaluation of natural antioxidant capacity/activity (IUPAC Technical Report). Pure and Applied Chemistry, 85(5), 957–998. https://doi.org/10.1351/PAC-REP-12-07-15

49. Speisky, H., Lopez-Alarcon, C., Gomez, M., Fuentes, J., Sandoval-Acuna, C. (2012). First web-based database on total phenolics and oxygen radical absorbance capacity (ORAC) of fruits produced and consumed within the south Andes region of South America. Journal of Agricultural and Food Chemistry, 60(36), 8851–8859. https://doi.org/10.1021/jf205167k

50. Atala, E., Vásquez, L., Speisky, H., Lissi, E., López-Alarcón, C. (2009). Ascorbic acid contribution to ORAC values in berry extracts: An evaluation by the ORAC pyrogallol red methodology. Food Chemistry, 113(1), 331–335. https://doi.org/10.1016/j.foodchem.2008.07.063

51. Ninfali, P., Chiarabini, A., Angelino, D. (2014). The ORAC/kcal ratio qualifies nutritional and functional properties of fruit juices, nectars, and fruit drinks. International Journal of Food Sciences and Nutrition, 65(6), 708–712. https://doi.org/10.3109/09637486.2014.918591

52. Atala, E., Aspée, A., Speisky, H., Lissi, E., López-Alarcón, C. (2013). Antioxidant capacity of phenolic compounds in acidic medium: A pyrogallol red-based ORAC (oxygen radical absorbance capacity) assay. Journal of Food Composition and Analysis, 32(2), 116–125. https://doi.org/10.1016/j.jfca.2013.09.007

53. Yiasmin, N., Waleed, A. L.-A., (2021). Recent applications of HPLC in food analysis: A mini review. International Journal of Advanced Engineering, Management and Science, 7(5), 01–06. http://doi.org/10.22161/ijaems.75.1

54. Esmail, L. A., Jabbar, H. S. (2023). Encapsulation of amaranth CDs at ZIF 7 MOFs as a novel adsorbent for ultrasonic-assisted dispersive nano-solid-phase microextraction and ultrasensitive determination of Allura red in food samples. Microchemical Journal, 195, Article 109474. https://doi.org/10.1016/j.microc.2023.109474

55. Baruah, B., Gabriel, G. J., Akbashev, M. J., Booher, M. E. (2013). Facile synthesis of silver nanoparticles stabilized by cationic polynorbornenes and their catalytic activity in 4-nitrophenol reduction. Langmuir, 29(13), 4225–4234. https://doi.org/10.1021/la305068p

56. Malik, A., Nath, M. (2020). Synthesis of Ag/ZIF 7 by immobilization of Ag nanoparticles onto ZIF 7 microcrystals: A heterogeneous catalyst for the reduction of nitroaromatic compounds and organic dyes. Journal of Environmental Chemical Engineering, 8(6), Article 104547. https://doi.org/10.1016/j.jece.2020.104547

57. González, C., Astudillo, C. A., López-Cortés, X. A., Maldonado, S. (2023). Semisupervised learning for MALDI–TOF mass spectrometry data classification: An application in the salmon industry. Neural Computing and Applications, 35, 9381–9391. https://doi.org/10.1007/s00521-023-08333-2

58. Mandal, S. M., Dey, S. (2008). LC–MALDI-TOF MS based rapid identification of phenolic acids. Journal of Biomolecular Techniques, 19(2), 116–121.

59. Wang, J., Kalt, W., Sporns, P. (2000). Comparison between PLC and MALDI-TOF MS analysis of anthocyanins in highbush blueberries. Journal of Agricultural and Food Chemistry, 48(8), 3330–3335. https://doi.org/10.1021/jf000101g

60. Wołosiak, R., Drużyńska, B., Derewiaka, D., Piecyk, M., Majewska, E., Ciecierska, M. et al. (2022). Verification of the conditions for determination of antioxidant activity by ABTS and DPPH assays — A practical approach. Molecules, 27(1), Article 50. https://doi.org/10.3390/molecules27010050

61. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26(9–10), 1231–1237. https://doi.org/10.1016/S0891-5849(98)00315-3

62. Brand-Williams, W., Cuvelier, M. E., Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT — Food Science and Technology, 28(1), 25–30. https://doi.org/10.1016/S0023-6438(95)80008-5

63. Brusotti, G., Cesari, I., Dentamaro, A., Caccialanza, G., Massolini, G. (2014). Isolation and characterization of bioactive compounds from plant resources: The role of analysis in the ethnopharmacological approach. Journal of Pharmaceutical and Biomedical Analysis, 87, 218–228. https://doi.org/10.1016/j.jpba.2013.03.007

64. Tzanova, M., Atanasov, V., Yaneva, Z., Ivanova, D., Dinev, T. (2020). Selectivity of current extraction techniques for flavonoids from plant materials. Processes, 8(10), Article 1222. https://doi.org/10.3390/pr8101222

65. Alekseenko, E. V., Bakumenko, O. E., Azarova, M. M., Isabayev, I. B., Kurbanov, M. T. (2019). The influence of pre-processing of berries cranberries on the extraction of anthocyanin pigments, the yield of juice and its antioxidant activity. Storage and Processing of Farm Products, 4, 10–27. (In Russian) https://doi.org/10.36107/spfp.2019.200